The rapid loss of cytochrome P-450 mediated drug metabolizing activities has greatly impended the use of cultured rat hepatocytes in studies of drug induced hepatotoxicity. Recent improvements in culture conditions have enabled cytochrome P-450 to be maintained for 24 hr. We have confirmed these studies and have investigated the metabolism and toxicity of acetaminophen after various periods of culture. Three major metabolites of acetaminophen have been detected in the culture medium; glucuronide, sulphate and glutathione conjugates. Changes in the relative production of these three conjugates in cultures occurred after 1 day suggesting adaptation to the culture conditions. Experiments were performed with hepatocyes prepared from animals induced with phenobarbital in vivo. These induced cells maintained high levels of cytochrome P-450 after 24 hr. and metabolized acetaminophen at rates which were similar but not identical to those found with fresh cells and exhibited toxicity within 6 hr. Thus, hepatocytes cultured for 24 hr. appear to be useful for studying acetaminophen and other drug-induced toxicities.